Colicin E9

Colicin E9 is a type of Colicin, an approximately 60kDa bacteriocin made by E. coli which acts against other nearby E. coli to kill them with its DNase activity; it digests the host's genome at specific locations, ultimately leading to the death of the cell.

Synthesis and release
Colicin E9 in solution, ie in the cytoplasm after synthesis, is monomeric, and forms a high affinity complex with its immunity protein, Im9. The immunity protein does not directly bind to the active site, but instead to an exosite. This is bound while in the producing cell to protect it from the activity. The structure shown is that of the DNase domain of colicin E9 bound to Im9.

Mechanism of uptake
The primary receptor for colicin E9 is the vitamin B12 receptor, BtuB. It then requires the outer membrane porin OmpF - either the two form the functional receptor, or OmpF is recruited for subsequent translocation. The OmpF association with the BtuB-colicin complex is weak and transient. After the interaction with OmpF, colicin E9 requires the TolB system to pass across the periplasm. The interaction with TolB is governed by a pentapeptide region in the N terminus called the TolB box, where ColE9 folds into a distorted hairpin within the six-bladed &beta;-propeller of TolB. The residues surrounding these (from 34 to 46) are unstructured and highly flexible, but the TolB box of 5 residues (DGSGW) is organised within this disordered domain. Within this pentapeptide sequence, the 3 essential resides are D35, S37 and W39. Mutations in all but one of these residues leads to a reduced affinity of binding to TolB. Some regions in the entire site have reduced mobility relative to other regions, that form local hydrophobic clusters.

OmpF acts synergistically with BtuB to protect bacteria against the action of colicin E9. This could indicate that OmpF is a component of the receptor apparatus. Alternatively the role of OmpF could be more to do with translocation rather than receptor recognition .

Once bound to the BtuB receptor, it is suggested that the coiled-coil receptor binding domain of the colicin unfolds to lose the immunity protein, Im9, and trigger translocation. This flexibility is crucial for translocation, and therefore the cytotoxicity, as shown by the addition of disulphide bonds. This reduces the flexibility and lowers the activity.

The endonuclease domain of colicin E9 is able to form ion channels in planar lipid bilayers. The E9 DNase mediates its own translocation across the cytoplasmic membrane, and the formation of ion channels is essential to this process. The association of colicin E9 with negative phospholipids results in a destabilisation of the DNase. This is protected by the colE9 immunity protein, Im9, but not by the binding of zinc to the active site. Formation of this destabilising complex preempts channel formation by the DNase, and makes up the first step in the translocation of colE9 across the E. coli inner membrane. The channels are then assumed to reseal themselves once the cytotoxic domain of the colicin has entered the cytoplasm.

The destabilisation of the DNase domain upon interaction with negative phospholipids increases its susceptibility to proteolysis and to thermal and chemical denaturation. Once associated, there is a massive disruption of protein tertiary structure, and the secondary structure instead interacts with the lipid bilayer - similar to the interaction between domains involved in Pore Formation in other colicins and the membranes that they disrupt.

The formation of a disulphide bond at D20C/E66C abolishes its channel forming ability, and its cytotoxicity (as it cannot penetrate cells) but has no effect on its DNase activity. It is still able to bind to the phospholipids, but not translocate across the membrane.

This uptake is highly similar to the uptake of Colicin E3.

Killing Activities
The cytotoxic activity of colE9 is DNase activity in the 15kDa C terminal domain, where it hydrolyses the DNA. However, it is also able to form ion channels in planar lipid bilayers, similar to the pore-forming colicins. These channels do not cause cell death, instead they are related to the ability of the E9 DNase domain to translocate across the inner membrane. The structure shows the crystal structure of the DNase domain.

The DNase domain nicks dsDNA at thymine bases.

The catalytic centre of the DNase domain contains the H-N-H motif, a site for DNA and metal binding. ColE9 binds Mg2+ as its cofactor. Binding the magnesium ion stabilises the protein, and regulates the binding of phosphate ions to the active site. Upon binding to the ion, the conformation of the DNase alters. This diagram shows the HNH motif found in ColE9.



In response to the DNA damage by colE9, the E. coli cell initiates an SOS response, prior to cell death. This involves the strong induction of 28 genes of the LexA-regulated SOS response.